First Regioselective Enzymatic Alkoxycarbonylation of Primary Amines. Synthesis of Novel 5′- and 3′-Carbamates of Pyrimidine 3′,5′-Diaminonucleoside Derivatives Including BVDU Analogues

Article abstract of DOI:10.1021/jo035678m

The first regioselective enzymatic alkoxycarbonylation of primary amino groups has been achieved in pyrimidine 3′,5′-diaminonucleoside derivatives. Thus, Candida antarctica lipase B (CAL-B) catalyzed this reaction with nonactivated homocarbonates allowing

Full text of DOI:10.1021/jo035678m

vated homocarbonates for preparing chiral protected
amines with allyl and benzyloxycarbonyl groups.
Enzymatic reactions have been important processes for
achieving nucleoside analogues, relevant compounds due
to their inherent value as potential therapeutical agents.9
In particular, pyrimidine nucleoside analogues have
shown remarkable antiviral10 and antitumor11 activities.
For example, (E)-5-(2-bromovinyl)-2′-deoxyuridine (BVDU)
has been one of them for treating Herpes simplex virus
type 1 (HSV-1) and Varicella zoster virus (VZV).12 In
recent years, the search for new nucleoside derivatives
using this clean, simple, and efficient methodology has
received a great deal of attention from organic chemists.13
In this context, oxime carbonates have been used with
lipases for direct and selective protection of natural
nucleosides.6
This regioselective process would be more difficult in
the case of two primary amines due to the major
nucleophilic character of the amino group compared to
the hydroxyl group. In fact, to the best of our knowledge,
there is no example of a regioselective enzymatic alkoxy-
carbonylation in amines. Recently, we have developed the
regioselective acylation of 3′,5′-diaminonucleosides with
lipases and nonactivated esters.14 In this paper, we report
the synthesis of novel pyrimidine aminonucleosides re-
gioselectively protected as carbamates. For that, direct
enzymatic alkoxycarbonylation reaction with Candida
antarctica lipase B (CAL-B) was carried out to synthesize
N-5′ carbamates, whereas the N-3′ regioisomers were
prepared using a short and efficient chemoenzymatic
route.
First, pyrimidine diaminonucleosides were synthesized
through efficient routes previously described for us.14 For
the alkoxycarbonylation reaction, nonactivated carbon-
ates were used as carbonylating agents since amines are
much more nucleophilic than alcohols and oxime carbon-
ates react nonenzymatically with the substrates. Specif-
ically, homocarbonates were used because their sym-
metrical structure gives a single unambiguous product.
For 3′,5′-diamino-2′,3′,5′-trideoxyuridine (1, Scheme 1),
a mixture of THF/Py was chosen to dissolve it, whereas
for 3′,5′-diamino-3′,5′-dideoxythymidine (2) and (E)-3′,5′-
diamino-5-(2-bromovinyl)-2′,3′,5′-trideoxyuridine (3), THF
was used as solvent. The enzymatic reactions were
carried out in the presence of molecular sieves (4 Å),
F ir st Regioselective En zym a tic
Alk oxyca r bon yla tion of P r im a r y Am in es.
Syn th esis of Novel 5′- a n d 3′-Ca r ba m a tes of
P yr im id in e 3′,5′-Dia m in on u cleosid e
Der iva tives In clu d in g BVDU An a logu es
Iva´n Lavandera, Susana Ferna´ndez,
Miguel Ferrero, and Vicente Gotor*
Departamento de Qu´ımica Orga´nica e Inorga´nica, Facultad
de Quı´mica, Universidad de Oviedo, 33071-Oviedo, Spain
vgs@fq.uniovi.es
Received November 14, 2003
Abstr a ct: The first regioselective enzymatic alkoxycarbo-
nylation of primary amino groups has been achieved in
pyrimidine 3′,5′-diaminonucleoside derivatives. Thus, Can-
dida antarctica lipase B (CAL-B) catalyzed this reaction with
nonactivated homocarbonates allowing the selective synthe-
sis of several N-5′ carbamates, including (E)-5-(2-bromovi-
nyl)-2′-deoxyuridine (BVDU) analogues, with moderate-high
yields, whereas immobilized Pseudomonas cepacia lipase
(PSL-C) afforded mixtures of alkoxycarbonylated regioiso-
mers. To obtain N-3′ carbamates selectively, a short and
efficient chemoenzymatic route was used employing some
of the N-5′-protected derivatives previously synthesized.
Lipases are one of the most used biocatalysts due to
their versatility in accepting a wide range of nucleophiles
(alcohols, amines, thiols, water, etc.) and carbonylating
agents (esters, anhydrides, carbonates, etc.).1 The enzy-
matic alkoxycarbonylation reaction has been scarcely
studied,2 despite the biological relevance shown by
carbonates and carbamates.3 In this field, our research
group has contributed to achieving the first example of
alkoxycarbonylation of amines4 and the regioselective
synthesis of carbonates in carbohydrates,5 nucleosides,6
and 1R,25-dihydroxyvitamin D3 A-ring precursors7 using
vinyl or oxime carbonates. It is noteworthy that through
these processes it is possible to introduce functionalities
selectively which act as protected or activated groups in
alcohols and amines. In the last case, reactions are
irreversible since carbamates are not substrates to li-
pases. Thus, Wong and co-workers8 have used nonacti-
(1) (a) Faber, K. Biotransformations in Organic Chemistry, 4th ed.;
Springer: Berlin, 2000. (b) Bornscheuer, U. T.; Kazlauskas, R. J .
Hydrolases in Organic Synthesis; Weinheim: Wiley-VCH: 1999.
(2) Gotor, V. Bioorg. Med. Chem. Lett. 1999, 7, 2189-2197.
(3) Parrish, J . P.; Salvatore, R. N.; J ung, K. W. Tetrahedron 2000,
56, 8207-8237.
(9) (a) Galmarini, C. M. Electron. J . Oncol. 2002, 1, 22-32. (b)
Robins, R. K.; Revankar, G. R. In Antiviral Drug Development; De
Clercq, E., Walker, R. T., Eds.; Plenum Press: New York, 1998; p 11.
(c) Mansour, T. S.; Storer, R. Curr. Pharm. Des. 1997, 3, 227-264.
(10) (a) Czernecki, S.; Valery, J . M. In Carbohydrates in Drug
Design; Witczak, Z. J ., Nieforth, K. A., Eds.; Dekker: New York, 1997;
p 495. (b) De Clercq, E. Pyrimidine Nucleoside Analogues as Antiviral
Agents. NATO ASI Ser., Ser. A 1984; 73, 203-230.
(4) Pozo, M.; Pulido, R.; Gotor, V. Tetrahedron 1992, 48, 6477-6484.
(5) (a) Pulido, R.; Gotor, V. Carbohydr. Res. 1994, 252, 55-68. (b)
Pulido, R.; Gotor, V. J . Chem. Soc., Perkin Trans. 1 1993, 589-592.
(6) (a) Mor´ıs, F.; Gotor, V. J . Org. Chem. 1992, 57, 2490-2492. (b)
Mor´ıs, F.; Gotor, V. Tetrahedron 1992, 48, 9869-9876.
(7) (a) D´ıaz, M.; Gotor-Ferna´ndez, V.; Ferrero, M.; Ferna´ndez, S.;
Gotor, V. J . Org. Chem. 2001, 66, 4227-4232. (b) Gotor-Ferna´ndez,
V.; Ferrero, M.; Ferna´ndez, S.; Gotor, V. J . Org. Chem. 1999, 64, 7504-
7510. (c) Ferrero, M.; Ferna´ndez, S.; Gotor, V. J . Org. Chem. 1997,
62, 4358-4363.
(11) Plunkett, W.; Gandhi, V. Cancer Chemother. Biol. Response
Modif. Annu. 2001, 19, 21-45.
(12) (a) De Clercq, E. Biochem. Pharmacol. 1984, 33, 2159-2169.
(b) De Clercq, E.; Descamps, J .; De Somer, P.; Barr, P. J .; J ones, A. S.;
Walker, R. T. Proc. Natl. Acad. Sci. U.S.A. 1979, 76, 2947-2951.
(13) (a) Ferrero, M.; Gotor, V. Chem. Rev. 2000, 100, 4319-4347.
(b) Ferrero, M.; Gotor V. Monatsh. Chem. 2000, 131, 585-616.
(14) (a) Lavandera, I.; Ferna´ndez, S.; Ferrero, M.; De Clercq, E.;
Gotor, V. Nucleosides Nucleotides Nucleic Acids 2003, 22, 1939-1952.
(b) Lavandera, I.; Ferna´ndez, S.; Ferrero, M.; Gotor, V. J . Org. Chem.
2001, 66, 4079-4082.
(8) (a) Takayama, S.; Lee, S. T.; Hung, S.-C.; Wong, C.-H. Chem.
Commun. 1999, 127-128. (b) Orsat, B.; Alper, P. B.; Moree, W.; Mak,
C.-P.; Wong, C.-H. J . Am. Chem. Soc. 1996, 118, 712-713.
10.1021/jo035678m CCC: $27.50 © 2004 American Chemical Society
Published on Web 02/03/2004
1748
J . Org. Chem. 2004, 69, 1748-1751

SCHEME 1
SCHEME 2
TABLE 1. Regioselective En zym a tic
Alk oxyca r bon yla tion of 3′,5′-Dia m in on u cleosid es w ith
CAL-B
TABLE 2. Regioselective En zym a tic
Alk oxyca r bon yla tion of 3′,5′-Dia m in on u cleosid es w ith
P SL-C
isol
alkoxycarbonylating
agent
yields
of N-5′
(%)a
alkoxycarbonylating
agent
isolated
yields (%)
sub-
entry strate
T
t
sub-
entry strate
t
R1
equiv solvent (°C) (h)
R1
equiv solvent
(h) N-3′ N-5′
1
2
3
4
5
6
7
8
9
1
1
1
2
2
2
3
3
3
Et
10
8
20
10
8
20
10
8
THF/Pyb 50
THF/Pyb 40
72
72
67
1
2
3
4
5
6
1
1
2
2
3
3
Et
50
40
50
40
50
40
THF/Pya 176 27
50
42
20b
56
CH2dCHCH2
Bn
Et
CH2dCHCH2
Bn
Et
69
CH2dCHCH2
Et
CH2dCHCH2
Et
THF/Pya 117 13
THF/Pyb 60 168
45c
62
THF
THF
THF
THF
30 22b
30 26
THF
THF
THF
THF
THF
THF
50
40
60 174
50 135
40
60 180
42
24
64
135 31b,c
96 24
50c
59
CH2dCHCH2
62
CH2dCHCH2
Bn
96
69
a
b
Ratio THF/Py 4.5:1 (v/v). Starting material was recovered.
50c
c Isolated as a mixture of products 6a /9a 1:1 by 1H NMR.
20
a
b
Isolated yields by flash chromatography. Ratio THF/Py 4.5:1
(v/v). c In addition to N-5′-alkoxycarbonylated derivative starting
of the more reactive diallyl carbonate at 40 °C were used
to alkoxycarbonylate with total regioselectivity 1 at the
5′ position, yielding 4b with 69% (entry 2, Table 1). On
the other hand, when dibenzyl carbonate was used, it was
necessary to increase the carbonate ratio to 20 equiv and
the temperature up to 60 °C due to the lower reactivity
of this agent.17 After 7 days, the N-Cbz-derivative 4c was
isolated exclusively with 45% yield, recovering a sub-
stantial amount of unreacted starting material (entry 3,
Table 1).
When similar processes were carried out with thymi-
dine and (E)-5-(2-bromovinyl)-2′-deoxyuridine derivatives
2 and 3, respectively, comparable behavior was observed.
CAL-B showed total regioselectivity toward the 5′-NH2,
isolating exclusively compounds 5 and 6 with moderately
high yields (entries 4-9, Table 1) and recovering part of
unreacted diaminonucleosides with dibenzyl carbonate
as carbonylating agent.
Then, we tried to find a biocatalyst which alkoxycar-
bonylated the amino group in the 3′ position. For that,
immobilized Pseudomonas cepacia lipase (PSL-C), which
has shown an excellent regioselectivity in the acetylation
of 3′-NH2 in 3′,5′-diaminonucleosides, was chosen (Scheme
2).14 The results are summarized in Table 2.
Unfortunately, PSL-C did not exhibit selectivity in the
alkoxycarbonylation of 1 with 50 equiv of diethyl carbon-
ate at 60 °C affording a mixture of both regioisomers 4a
and 7a (entry 1, Table 2). Compound 7a was identified
from its 1H NMR (D2O) spectrum, which presents a
downfield shift of the H3′ (from 3.33 ppm in 1 to 4.21 ppm
in 7a ), while H5′ almost did not change. Moreover,
experiments 2D HMBC showed a cross-peak between H3′
and CdO corresponding to correlation H3′-CNCO via
material was recovered.
which catalyzed these processes.15 To reach high conver-
sions, ratios of alkoxycarbonylating agents have been
established.
We focused on CAL-B as biocatalyst since this enzyme
has demonstrated an excellent regioselectivity toward the
amino group at the 5′ position in the enzymatic acylation
of diaminonucleosides.14 The study begins with the di-
amino derivative of 2′-deoxyuridine (1). Thus, when the
reaction was carried out with 10 equiv of diethyl carbon-
ate at 50 °C, the formation of a single product was
observed (monitored by TLC), which corresponded to
monoalkoxycarbonylated derivative at N-5′, 4a being
isolated with 67% yield after flash chromatography (entry
1, Table 1). We also confirmed that no reaction occurred
in the absence of the enzyme.16 The structure of this
1
compound was ascertained from its H NMR (MeOH-d4)
spectrum, which showed a downfield shift corresponding
to both H5′ protons from 2.8 to 3.07 ppm in diamino-
nucleoside 1 to 3.63 ppm in 4a . Furthermore, H3′ did not
display any significant change. In addition, heteronuclear
correlation 1H-13C experiments 2D HMBC showed a
cross-peak between H5′ and CdO, which corresponds to
3
correlation H5′-CNCO via J CH. It is noteworthy that
neither N-3′,5′- nor N-3′-alkoxycarbonylated derivatives
were formed, despite the fact that both amino groups are
primaries.
To confer versatility to this enzymatic reaction, other
alkoxycarbonyl moieties were introduced. Thus, 8 equiv
(15) To see an explanation for this effect: Lavandera, I.; Ferna´ndez,
S.; Ferrero, M.; Gotor, V. Tetrahedron 2003, 59, 5449-5456.
(16) Detailed conditions: corresponding diaminonucleosides 1-3
and homocarbonates were dissolved in THF/Py (for 1) or THF (for 2
and 3) at 60 °C during 8 days and no reactions were observed.
(17) The low reactivity of this carbonate has already been reported
in the ref 8.
J . Org. Chem, Vol. 69, No. 5, 2004 1749

SCHEME 3. Syn th esis of 3′-Ca r ba m a te Nu cleosid e
zyloxycarbonyl protecting groups, very useful for further
modifications, have also been introduced using this
enzymatic methodology. The utility of these modified
compounds have been presented, achieving the first
synthesis of N-3′-Cbz-3′,5′-diaminonucleoside analogues
through a short and efficient chemoenzymatic route with
high overall yield. It is noteworthy that a new family
derived from BVDU has been obtained. Preliminary
studies with some of these carbamate derivatives have
been performed against several viruses.19 They show
better results than the unmodified 3′,5′-diaminonucleo-
sides.20 The complete study of the biological activity of
these new derivatives will be reported in due course.
Der iva tivesa
a
Reaction conditions: (a) BnOCOCl, Na2CO3, THF/H2O, rt, 24
Exp er im en ta l Section
h; (b) PdCl2(PPh3)2, Bu3SnH, AcOH, CH2Cl2, rt, 4 h.
Gen er a l Meth od s. Candida antarctica lipase B (CAL-B,
Novozym 435, 7300 PLU/g) was a gift from Novo Nordisk Co.
Immobilized Pseudomonas cepacia lipase on ceramic particles
(PSL-C, 783 U/g) was purchased from Amano Pharmaceutical
Co.
3J CH. To avoid the formation of N-5′ monocarbonate 4a ,
lesser equivalents of diethyl carbonate and/or lower
temperatures were used, but in all cases a large amount
of starting nucleoside was recovered. With 40 equiv of
diallyl carbonate the process takes place with low selec-
tivity, obtaining mainly 4b (entry 2, Table 2).
Similar results were observed when substrates 2 and
3 were subjected to alkoxycarbonylation with PSL-C
(entries 3-6, Table 2). A mixture of both N-3′- and N-5′-
alkoxycarbonylated regioisomers was obtained, the latter
being the main product when diallyl carbonate was
utilized. In the case of dibenzyl carbonate, which had
already shown low reactivity with CAL-B, the reaction
did not occur.
Since direct enzymatic methodology did not allow for
N-3′-alkoxycarbonylated nucleoside derivatives with good
yields, an orthogonal protection scheme was designed.
The strategy starts with the protection of the 5′-NH2
group, subsequent derivatization of the N-3′ amine as a
carbamate, and finally selective deprotection of the 5′
position.
We decided to synthesize the N-3′-Cbz derivatives 7c-
9c (Scheme 3) due to the ease of removing this protecting
group for further modifications. To selectively protect the
5′-position, reaction of 1 with trityl chloride, a reagent
with a very appropriate bulky group to modify that
position in natural nucleosides was carried out. However,
a mixture of regioisomers was obtained due to the major
nucleophilic character of the amino groups.
We made use of the regioselective enzymatic alkoxy-
carbonylation shown previously to selectively protect the
5′-NH2 group as allyloxycarbamate affording derivatives
4b-6b. Treatment of the later compounds with benzy-
loxycarbonyl chloride at room temperature afforded di-
carbamate nucleosides 10-12 with high efficiency. Then,
selective deprotection of the allyloxycarbonyl group in the
conditions described by Guibe´ and co-workers18 gave
place to N-3′-Cbz-protected derivatives 7c-9c through
a short and efficient chemoenzymatic route.
Regioselective enzymatic alkoxycarbonylation of amines
using a very simple procedure with nonactivated ho-
mocarbonates and CAL-B as biocatalyst have been shown
for the first time, synthesizing N-5′-carbamate pyrimi-
dine 3′,5′-diaminonucleoside derivatives. Allyloxy 8 ben-
Gen er a l P r oced u r e for th e En zym a tic Alk oxyca r bon y-
la tion of 3′,5′-Dia m in on u cleosid es. Corresponding homocar-
bonate (diethyl, diallyl, or dibenzyl carbonates) was added to a
suspension of diaminonucleoside (20 mg, in the case of 1 it was
previously dissolved in 1 mL of dry pyridine), lipase (10 mg of
CAL-B or 130 mg of PSL-C), and molecular sieves 4 Å (20 mg)
in dry THF (4.5 mL) under nitrogen, and the mixture was stirred
at 250 rpm (ratios, temperatures, and reaction times are
indicated in Tables 1 and 2). Then, the enzyme and molecular
sieves were filtered off and washed with MeOH (3 × 5 mL). The
filtrate was evaporated to dryness, and the crude residue was
purified by flash chromatography (gradient eluent 10% MeOH/
EtOAc-MeOH for compounds 4a -c, 5a -c, and 6a -c and
gradient eluent 10% MeOH/EtOAc-MeOH-10% NH3(aq)/MeOH
for compounds 7a ,b, 8a ,b, and 9a ,b).
Syn th esis of Dica r ba m a te Nu cleosid es 10-12. To a
solution of N-5′-protected nucleosides 4b-6b (20 mg, 1 equiv)
in a mixture of H2O (1 mL) and THF (0.5 mL) were added sodium
carbonate (1.2 equiv) and benzyloxycarbonyl chloride (1.2 equiv).
The mixture was stirred at room temperature during 24 h. The
solvents were evaporated under vacuum, and the crude residue
was purified by flash chromatography (gradient eluent 20%
hexane/EtOAc-EtOAc).
Syn th esis of Mon oca r ba m a te Nu cleosid es 7c-9c. Sub-
strates 10-12 (20 mg, 1 equiv), PdCl2(PPh3)2 (0.02 equiv), and
acetic acid (2.4 equiv) were dissolved in dry CH2Cl2 under
nitrogen. With a syringe, Bu3SnH (1.1 equiv) was then added
rapidly in one portion. The reaction was stirred at room
temperature during 4 h. The solvent was evaporated under
vacuum, and the crude residue was purified by flash chroma-
tography (gradient eluent MeOH-5% NH3(aq)/MeOH).
3′-Am in o-5′-et h oxyca r b on yla m in o-2′,3′,5′-t r id eoxyu r i-
d in e (4a ): 1H NMR (MeOH-d4, 200 MHz) δ 1.43 (t, 3H, H4′′
,
3J HH ) 7.1 Hz), 2.36-2.49 (m, 2H, H2′), 3.50 (m, 1H, H3′), 3.63
3
(m, 2H, H5′), 3.89 (m, 1H, H4′), 4.27 (q, 2H, H3′′, J HH ) 7.1 Hz),
5.88 (d, 1H, H5, 3J HH ) 8.1 Hz), 6.29 (dd, 1H, H1′, 3J HH ) 6.8 Hz,
3
3J HH ) 5.1 Hz), 7.92 (d, 1H, H6, J HH ) 8.1 Hz); MS (ESI+, m/z)
337 [(M + K)+, 8], 321 [(M + Na)+, 100], 299 [(M + H)+, 31].
5′-Allyloxyca r bon yla m in o-3′-a m in o-2′,3′,5′-tr id eoxyu r i-
d in e (4b): 1H NMR (MeOH-d4, 300 MHz) δ 2.36-2.53 (m, 2H,
H2′), 3.50 (m, 1H, H3′), 3.59-3.72 (m, 2H, H5′), 3.90 (m, 1H, H4′),
3
3
4.74 (d, 2H, H3′′, J HH ) 5.4 Hz), 5.37 (dd, 1H, H5′′Z, J HH ) 10.5
2
2
| J HH| 1.4 Hz), 5.50 (dd, 1H, H5′′E
,
3J HH ) 17.1 | J HH| 1.4 Hz),
3
5.87 (d, 1H, H5, J HH ) 8.0 Hz), 6.06-6.19 (m, 1H, H4′′), 6.29
3
3
(dd, 1H, H1′, J HH ) 6.8 Hz, J HH ) 4.6 Hz), 7.92 (d, 1H, H6,
(19) The antiviral assays are being performed in the Rega Institute
for Medical Research (Lovaina, Belgium) by Prof. Erik De Clercq.
(20) Lavandera, I.; Ferna´ndez, S.; Ferrero, M.; De Clercq, E.; Gotor,
V. Nucleosides Nucleotides Nucleic Acids 2003, 22, 833-836.
(18) Dangles, O.; Guibe´, F.; Balavoine, G.; Lavielle, S.; Marquet, A.
J . Org. Chem. 1987, 52, 4984-4993.
1750 J . Org. Chem., Vol. 69, No. 5, 2004

3J HH ) 8.0 Hz); MS (ESI+, m/z) 349 [(M + K)+, 20], 333 [(M +
Na)+, 100], 311 [(M + H)+, 42].
7.53 (br s, 5H, Har), 7.73 (s, 1H, H6); MS (ESI+, m/z) 397 [(M +
Na)+, 9], 375 [(M + H)+, 63].
3′-Am in o-5′-ben zyloxycar bon ylam in o-2′,3′,5′-tr ideoxyu r i-
d in e (4c): 1H NMR (MeOH-d4, 200 MHz) δ 2.41 (m, 2H, H2′),
3.48 (m, 1H, H3′), 3.67 (m, 2H, H5′), 3.88 (m, 1H, H4′), 5.21-5.36
(E)-5′-Am in o-3′-ben zyloxyca r bon yla m in o-5-(2-br om ovi-
n yl)-2′,3′,5′-tr id eoxyu r id in e (9c): 1H NMR (MeOH-d4, 300
MHz) δ 2.55-2.72 (m, 2H, H2′), 3.27-3.42 (m, 2H, H5′), 4.08 (m,
1H, H4′), 4.44 (m, 1H, H3′), 5.29 (br s, 2H, H3′′), 6.28 (dd, 1H, H1′,
3
(m, 2H, H3′′), 5.79 (d, 1H, H5, J HH ) 8.1 Hz), 6.27 (dd, 1H, H1′,
3
3
3J HH ) 6.3 Hz, J HH ) 4.9 Hz), 7.48-7.55 (m, 5H, Har), 7.87 (d,
3J HH ) 5.7 Hz), 7.05 (d, 1H, H7, J HH 13.7 Hz), 7.53 (m, 6H, Har
3
1H, H6, J HH ) 8.1 Hz); MS (ESI+, m/z) 399 [(M + K)+, 19], 383
+ H8), 7.98 (s, 1H, H6); MS (ESI+, m/z) 505 [(M81Br + K)+, 3],
503 [(M79Br + K)+, 3], 489 [(M81Br + Na)+, 30], 487 [(M79Br +
Na)+, 28], 467 [(M81Br + H)+, 100], 465 [(M79Br + H)+, 99].
5′-Allyloxyca r bon yla m in o-3′-ben zyloxyca r bon yla m in o-
2′,3′,5′-tr id eoxyu r id in e (10):21 1H NMR (CDCl3, 200 MHz) δ
2.26-2.39 (m, 2H, H2′), 3.39 (m, 1H, H5′), 3.62 (m, 1H, H5′), 3.89
[(M + Na)+, 100], 361 [(M + H)+, 41].
3′-Am in o-5′-et h oxyca r b on yla m in o-3′,5′-d id eoxyt h ym i-
d in e (5a ): 1H NMR (MeOH-d4, 200 MHz) δ 1.43 (t, 3H, H4′′
,
3J HH ) 7.1 Hz), 2.09 (s, 3H, H7), 2.42 (m, 2H, H2′), 3.55 (m, 1H,
H3′), 3.64 (m, 2H, H5′), 3.84 (m, 1H, H4′), 4.28 (q, 2H, H3′′,
3J HH
) 7.1 Hz), 6.31 (dd, 1H, H1′, 3J HH ) 6.8 Hz, 3J HH ) 4.9 Hz), 7.74
(s, 1H, H6); MS (ESI+, m/z) 351 [(M + K)+, 10], 335 [(M + Na)+,
100], 313 [(M + H)+, 28].
(m, 1H, H4′), 4.11 (m, 1H, H3′), 4.56 (d, 2H, H3′′, J HH ) 5.1 Hz),
3
5.11-5.33 (m, 4H, H3′′′ + H5′′Z + H5′′E), 5.73-6.04 (m, 4H, H5
+
H4′′ + 2 NH), 6.13 (dd, 1H, H1′, 3J HH ) 6.2 Hz), 7.34 (s, 5H, Har),
3
5′-Allyloxyca r bon yla m in o-3′-a m in o-3′,5′-d id eoxyth ym i-
d in e (5b): 1H NMR (MeOH-d4, 200 MHz) δ 2.08 (s, 3H, H7),
2.43 (m, 2H, H2′), 3.50 (m, 1H, H3′), 3.66 (m, 2H, H5′), 3.88 (m,
7.43 (d, 1H, H6, J HH ) 8.7 Hz), 9.75 (s, 1H, NH); MS (ESI+,
m/z) 483 [(M + K)+, 67], 467 [(M + Na)+, 100].
5′-Allyloxyca r bon yla m in o-3′-ben zyloxyca r bon yla m in o-
3′,5′-d id eoxyth ym id in e (11):21 1H NMR (CDCl3, 200 MHz) δ
1.90 (s, 3H, H7), 2.18-2.35 (m, 2H, H2′), 3.43 (m, 1H, H5′), 3.62
(m, 1H, H5′), 3.89 (m, 1H, H4′), 4.11 (m, 1H, H3′), 4.57 (d, 2H,
1H, H4′), 4.74 (d, 2H, H3′′
,
3J HH ) 5.1 Hz), 5.36 (dd, 1H, H5′′Z
,
|
2
2
3J HH ) 10.2 | J HH| 1.2 Hz), 5.49 (dd, 1H, H5′′E, 3J HH ) 17.3 | J HH
3
1.2 Hz), 6.03-6.21 (m, 1H, H4′′), 6.31 (dd, 1H, H1′, J HH ) 6.6
Hz), 7.73 (s, 1H, H6); MS (ESI+, m/z) 363 [(M + K)+, 5], 347 [(M
+ Na)+, 100], 325 [(M + H)+, 27].
H
3′′, 3J HH ) 5.1 Hz), 5.10-5.33 (m, 4H, H3′′′ + H5′′Z + H5′′E), 5.72
3
(br s, 1H, NH), 5.80-5.99 (m, 1H, H4′′), 6.20 (dd, 1H, H1′, J HH
3
3′-Am in o-5′-b en zyloxyca r b on yla m in o-3′,5′-d id eoxyt h y-
m id in e (5c): 1H NMR (MeOH-d4, 200 MHz) δ 2.00 (s, 3H, H7),
2.39 (m, 2H, H2′), 3.50 (m, 1H, H3′), 3.69 (m, 2H, H5′), 3.88 (m,
) 5.9 Hz), 6.28 (d, 1H, NH, J HH ) 6.4 Hz), 7.34 (m, 6H, Har +
H6), 9.77 (s, 1H, NH); MS (ESI+, m/z) 497 [(M + K)+, 100], 481
[(M + Na)+, 76].
3
1H, H4′), 5.21-5.37 (m, 2H, H3′′), 6.30 (dd, 1H, H1′, J HH ) 6.4
(E)-5′-Allyloxycar bon ylam in o-3′-ben zyloxycar bon ilam in o-
5-(2-br om ovin yl)-2′,3′,5′-tr id eoxyu r id in e (12):21 1H NMR
(MeOH-d4, 300 MHz) δ 2.55 (m, 2H, H2′), 3.57-3.74 (m, 2H, H5′),
4.08 (m, 1H, H4′), 4.31 (m, 1H, H3′), 4.75 (br s, 2H, H3′′), 5.28-
Hz, 3J HH ) 5.1 Hz), 7.46-7.53 (m, 5H, Har), 7.71 (s, 1H, H6); MS
(ESI+, m/z) 413 [(M + K)+, 14], 397 [(M + Na)+, 100], 375 [(M +
H)+, 63].
3
2
(E)-3′-Am in o-5-(2-br om ovin yl)-5′-eth oxyca r bon yla m in o-
5.37 (m, 3H, H3′′′ + H5′′Z), 5.49 (dd, 1H, H5′′E, J HH ) 17.3 | J HH
|
2′,3′,5′-tr id eoxyu r id in e (6a ): 1H NMR (MeOH-d4, 200 MHz)
1.4 Hz), 6.06-6.18 (m, 1H, H4′′), 6.30 (dd, 1H, H1′, J HH ) 6.0
3
3J HH ) 7.1 Hz), 2.45 (m, 2H, H2′), 3.50 (m,
Hz), 7.06 (d, 1H, H7, J HH ) 13.7 Hz), 7.54 (m, 6H, Har + H8),
3
δ 1.46 (t, 3H, H4′′
1H, H3′), 3.68 (m, 2H, H5′), 3.88 (m, 1H, H4′), 4.31 (q, 2H, H3′′
3J HH ) 7.1 Hz), 6.28 (dd, 1H, H1′, 3J HH ) 6.1 Hz, 3J HH ) 4.9 Hz),
,
,
7.99 (s, 1H, H6); MS (ESI+, m/z) 589 [(M81Br + K)+, 22], 587
[(M79Br + K)+, 30], 573 [(M81Br + Na)+, 100], 571 [(M79Br +
Na)+, 91], 551 [(M81Br + H)+, 7], 549 [(M79Br + H)+, 8].
3
3
7.07 (d, 1H, H7, J HH ) 13.7 Hz), 7.56 (d, 1H, H8, J HH ) 13.7
Hz), 8.01 (s, 1H, H6); MS (ESI+, m/z) 443 [(M81Br + K)+, 15],
441 [(M79Br + K)+, 13], 427 [(M81Br + Na)+, 100], 425 [(M79Br
+ Na)+, 98], 405 [(M81Br + H)+, 73], 403 [(M79Br + H)+, 75].
(E)-5′-Allyloxycar bon ylam in o-3′-am in o-5-(2-br om ovin yl)-
2′,3′,5′-tr id eoxyu r id in e (6b): 1H NMR (MeOH-d4, 200 MHz)
δ 2.44 (m, 2H, H2′), 3.50 (m, 1H, H3′), 3.68 (m, 2H, H5′), 3.92 (m,
Ack n ow led gm en t. We thank Prof. Erik De Clercq
for the biological assays of these compounds. We express
our appreciation to Novo Nordisk Co. for the generous
gift of the CAL-B. Financial support of this work by the
Spanish Ministerio de Ciencia y Tecnolog´ıa (Project
PPQ-2001-2683) and by Principado de Asturias (Project
GE-EXP01-03) is gratefully acknowledged. S.F. thanks
MCYT for a personal grant (Ramo´n y Cajal Project). I.L.
thanks MCYT for a pre-doctoral fellowship.
1H, H4′), 4.76 (d, 2H, H3′′
,
3J HH ) 5.6 Hz), 5.38 (dd, 1H, H5′′Z
,
|
2
2
3J HH ) 10.5 | J HH| 1.5 Hz), 5.50 (dd, 1H, H5′′E, 3J HH ) 17.3 | J HH
3
1.5 Hz), 6.05-6.22 (m, 1H, H4′′), 6.29 (dd, 1H, H1′, J HH ) 6.1
3
3
Hz), 7.06 (d, 1H, H7, J HH ) 13.7 Hz), 7.56 (d, 1H, H8, J HH
)
13.7 Hz), 8.01 (s, 1H, H6); MS (ESI+, m/z) 439 [(M81Br + Na)+,
97], 437 [(M79Br + Na)+, 100], 417 [(M81Br + H)+, 89], 415 [(M79
Br + H)+, 86].
-
Su p p or tin g In for m a tion Ava ila ble: Complete 1H, 13C,
and DEPT NMR spectral data and some 2D NMR experiments
are shown in addition to mp, IR, microanalysis, and MS data.
The level of purity is indicated by the inclusion of copies of 1H
and 13C NMR spectra. This material is available free of charge
via the Internet at http://pubs.acs.org.
(E)-3′-Am in o-5′-ben zyloxyca r bon yla m in o-5-(2-br om ovi-
n yl)-2′,3′,5′-tr id eoxyu r id in e (6c): 1H NMR (MeOH-d4, 300
MHz) δ 2.42 (m, 2H, H2′), 3.49 (m, 1H, H3′), 3.61-3.75 (m, 2H,
3
H5′), 3.90 (m, 1H, H4′), 5.31 (m, 2H, H3′′), 6.27 (dd, 1H, H1′, J HH
3
) 6.1 Hz), 7.05 (d, 1H, H7, J HH ) 13.6 Hz), 7.45-7.57 (m, 6H,
Har+H8), 8.00 (s, 1H, H6); MS (ESI+, m/z) 489 [(M81Br + Na)+,
32], 487 [(M79Br + Na)+, 33], 467 [(M81Br + H)+, 94], 465 [(M79
-
J O035678M
Br + H)+, 100].
5′-Am in o-3′-ben zyloxycar bon ylam in o-2′,3′,5′-tr ideoxyu r i-
d in e (7c): 1H NMR (MeOH-d4, 200 MHz) δ 2.55 (m, 2H, H2′),
3.10 (m, 2H, H5′), 3.92 (m, 1H, H4′), 4.35 (m, 1H, H3′), 5.27 (br s,
2H, H3′′), 5.90 (d, 1H, H5, 3J HH ) 8.3 Hz), 6.27 (dd, 1H, H1′, 3J HH
(21) Structures of compounds 10-12 are labeled as follows:
3
) 7.1 Hz), 7.53 (s, 5H, Har), 7.93 (d, 1H, H6, J HH ) 8.3 Hz); MS
(ESI+, m/z) 383 [(M + Na)+, 71], 361 [(M + H)+, 100].
5′-Am in o-3′-b en zyloxyca r b on yla m in o-3′,5′-d id eoxyt h y-
m id in e (8c): 1H NMR (MeOH-d4, 200 MHz) δ 2.09 (s, 3H, H7),
2.44-2.67 (m, 2H, H2′), 3.11 (m, 2H, H5′), 3.93 (m, 1H, H4′), 4.37
3
(m, 1H, H3′), 5.28 (s, 2H, H3′′), 6.33 (dd, 1H, H1′, J HH ) 5.6 Hz),
J . Org. Chem, Vol. 69, No. 5, 2004 1751